1. FIELD OF THE INVENTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
3.1. DEFINITIONS AND ABBREVIATIONS
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. OMP21 POLYPEPTIDE
5.2. OMP21-DERIVED POLYPEPTIDES
5.3. ISOLATION AND PURIFICATION OF OMP21
5.4. OMP21 IMMUNOGENS AND ANTI-OMP21 ANTIBODIES
5.5. PHARMACEUTICAL COMPOSITIONS
5.6. METHODS OF DETECTING
5.7. NUCLEIC ACIDS ENCODING OMP21
5.8. RECOMBINANT PRODUCTION OF OMP21
5.9. APPLICATIONS
6. EXAMPLE: ISOLATION AND CHARACTERIZATION OF THE OMP21 POLYPEPTIDE AND GENE ENCODING SAME
6.1. MATERIALS AND METHODS
6.1.1. DETERGENT EXTRACTION OF OMP21
6.1.2. AMINO TERMINAL SEQUENCING OF OMP21
6.1.3. ANTI-OMP21 ANTISERUM
6.1.4. WESTERN BLOTS
6.1.5. OUTER SURFACE LOCALIZATION OF OMP21
6.2. RESULTS
6.2.1. OUTER SURFACE LOCATION OF OMP21
6.2.2. PROPERTIES OF OMP21 POLYPEPTIDE
6.2.3. CONSERVATION OF OMP21 POLYPEPTIDE
7. EXAMPLE: EFFICACY OF OMP21 VACCINE: CYTOTOXIC ACTIVITY OF ANTI-OMP21 ANTISERUM
8. EXAMPLE: ISOLATION OF THE omp21 GENE
8.1. PREPARATION OF PRIMERS
8.2. SUPPRESSION PCR
8.3. ISOLATION AND SUBCLONING OF THE PCR PRODUCT
8.4. IDENTIFICATION OF RECOMBINANT PLASMIDS
8.5. SEQUENCE ANALYSIS
9. EXAMPLE: PREPARATION OF RECOMBINANT OMP21
9.1. CONSTRUCTION OF AN EXPRESSION VECTOR
9.2. EXPRESSION OF OMP21
10. EXAMPLE: VERIFICATION OF THE omp21 GENE
10.1. CONSTRUCTION OF AN omp21 GENE-TARGETING CASSETTE
10.2 PREPARATION OF COMPETENT MORAXELLA CATARRHALIS CELLS
10.3. ELECTROPORATION OF COMPETENT CELLS
10.4. PCR ANALYSIS OF PUTATIVE OMP21 DELETION MUTANTS
10.5. SOUTHERN ANALYSIS OF OMP 21 DELETION MUTANTS
11. EXAMPLE: RFLP ANALYSIS OF omp 21
12. EXAMPLE: GENERATION AND REACTIVITY OF MONOCLONAL ANTI-OMP21 ANTIBODIES
13. EXAMPLE: NASOPHARYNGEAL CELL BINDING
14. DEPOSIT OF MICROORGANISM
The present invention generally relates to an isolated or substantially purified protein obtainable from the outer membranes of M. catarrhalis, called xe2x80x9cOMP21xe2x80x9d (defined below in Section 3). The invention also encompasses the amino acid sequence thereof, and antibodies, including cytotoxic antibodies, that specifically bind OMP21. The invention further encompasses pharmaceutical compositions, including prophylactic or therapeutic compositions, and which may be immunogenic compositions, including vaccines. The invention additionally provides methods of preventing, treating or ameliorating disorders in mammals related to M. catarrhalis infections and for inducing immune responses to M. catarrhalis. The invention further provides isolated nucleotide sequences encoding the OMP21, homologous and complementary sequences thereto, vectors having said sequences, host cells containing said vectors, and prophylactic or therapeutic compositions, which may be immunogenic compositions, including vaccines comprising same. Diagnostic methods and kits are also included.
Moraxella catarrhalis, also known as Moraxella (Branhamella) catarrhalis or Branhamella catarrhalis and formerly known as Neisseria catarrhalis or Micrococcus catarrhalis, is a gram-negative bacterium frequently found in the respiratory tract of humans. M. catarrhalis, originally thought to be a harmless commensal organism, is now recognized as an important pathogen in upper and lower respiratory tract infections in humans. In humans, M. catarrhalis causes serious lower respiratory tract infections in adults with chronic lung disease, systemic infections in immunocompromised patients, and otitis media and sinusitis in infants and children (Helminen et al., 1993, Infect. Immun. 61:2003-2010; Catlin; B. W., 1990, Clin. Microbiol. Rev. 3:293-320; and references cited therein). The outer surface components of Moraxella catarrhalis have been studied in attempts to understand the pathogenic process of M. catarrhalis infections and to develop useful therapeutic treatments and prophylactic measures against such infections. The outer membrane proteins (OMPs) in particular have received considerable attention as possible virulence factors and as potential vaccine antigens. M. catarrhalis has over 20 different OMPs with 6 to 8 of these, OMPs A to H, as the predominate species (Murphy and Loeb, 1989, Microbial Pathogen. 6:159-174). The molecular weights of OMPs A to H range from 98 to 21 kD, respectively (Bartos and Murphy, 1988, J. Infect. Dis. 158:761-765; Helminen et al., 1993, Infect. Immun. 61:2003-2010; Murphy et al, 1993, Molecul. Microbiol. 10:87-97; and Sarwar et al, 1992, Infect. Immun. 60:804-809). Comparisons of protein profiles by sodium dodecylsulfate polyarylamide gel electrophoresis (SDS-PAGE) of outer membrane preparations from 50 M. catarrhalis strains show nearly homogeneous patterns of OMPs A to H (Bartos and Murphy, 1988, J. Infect. Dis. 158:761-765).
In intact bacterium or bacterially-derived outer membrane vesicles, several of the above-identified OMPs present surface-exposed epitopes that elicit the production of antibodies that bind the OMPs. These antigenic OMPs include OMP E and OMP G (Murphy and Bartos, 1989, Infect. Immun. 57:2938-2941); OMP C/D (Sarwar et al., 1992, Infect. Immun. 60:804-809); CopB, an 80 kD OMP, (Helminen et al., 1993, Infect. Immun. 61:2003-2010); and UspA (Helminen et al., 1994, J. Infect. Dis. 170:867-872).
The therapeutic potential of antibodies to surface-exposed epitopes of outer-membrane proteins of M. catarrhalis is generally examined by the cytotoxic (bactericidal) activity, because there is no animal model of disease. The only natural host for disease caused by Moraxella is human. However, others have studied the role of antibodies in an animal model of Moraxella lung clearance. The model involved direct bolus inoculation of lungs of BALB/c VAF/Plus mice with a controlled number of M. catarrhalis cells and subsequent examination of the rate of pulmonary clearance of the bacteria (Unhanand et al., 1992, J. Infect. Dis. 165:644-650). Different clinical isolates of the M. catarrhalis exhibited different rates of clearance, all of which are relatively rapid, that correlated with the level of granulocyte recruitment into the infection site. Passive immunization with a monoclonal antibody directed to a surface-exposed epitope of CopB and UspA increased the rate of pulmonary clearance of M. catarrhalis (Helminen et al., 1993, Infect. Immun. 61:2003-2010; Helminen et al., 1994, J. Infect. Dis. 170:867-872). There remains a need for compositions and methods for diagnosis of, as well as, prophylactic and therapeutic treatments for infections caused by M. catarrhalis. 
The adherence of bacterial pathogens to a host cell surface promotes colonization and initiates pathogenesis. See, E. H. Beachey, 1981, J. Infect. Dis. 143:325-345. Gram-negative bacteria typically express surface lectins that bind to specific oligosaccharides of glycoproteins and/or glycolipids on the host cell surface. Such lectins are often associated with pili or fimbriae. Bacterial adherence can also occur by non-specific binding resulting from hydrophobic and/or charge interaction with the host cell surface.
The mechanism of M. catarrhalis adherence to cells of the respiratory tract remains poorly understood. The organism adheres to cultured human nasopharyngeal epithelial cells. Another study suggests that fimbriae may have a role in the adherence to such cells as fimbriae denaturation or treatment with anti-fimbriae antibodies reduced adherence by fimbriated strains. Fimbriae mediated binding, however, cannot be the sole basis of this adherence as the most highly adhering strain, among the several examined, was a non-fimbriated strain. Thus, other unidentified components are involved in the bacteria""s adherence.
An object of the present invention is to provide an isolated or substantially purified OMP21 protein of a M. catarrhalis strain, wherein the apparent molecular weight is about 16 kD to about 20 kD, as predicted from the deduced amino acid sequence or determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (xe2x80x9cSDS-PAGExe2x80x9d). The term xe2x80x9cOMP21xe2x80x9d as used herein and in the claims is intended to globally encompass: all forms of the protein having molecular weight of 16 kd to 20 kd, including the native, wild-type OMP21 protein obtainable from M. catarrhalis, and xe2x80x9cOMP21-derived polypeptidesxe2x80x9d, as defined in Section 3.1 herein. Preferably, OMP21 has the sequence of any of SEQ ID Nos.: 1 or 7 or sequences substantially homologous thereto. More preferably, OMP21 is an outer membrane protein. Still more preferably, OMP21 has a nasopharyngeal binding domain.
It is intended that OMP21 obtainable from any commercially available strains and clinical isolates of Moraxella catarrhalis is included in this invention, however preferred is OMP21 obtainable from virulent clinical isolates. The OMP21 is at least 70 wt % pure, preferably at least about 90 wt % pure, and may be in the form of an aqueous solution thereof.
Another object of the present invention is to provide an isolated nucleic acid molecule encoding OMP21. Preferred is the nucleic acid sequence wherein the encoded OMP21 comprises the amino acid sequence of any of SEQ ID NOs.: 1 or 7 or sequences substantially homologous thereto. Also included is an isolated nucleic acid molecule comprising a sequence of any of SEQ ID NOs: 2-6 and 8-20, a complementary sequence thereof, sequences substantially homologous thereto, and any fragment thereof; a DNA sequence encoding a deduced amino acid sequence of any of SEQ ID Nos.:1 or 7, the complimentary sequence thereto, sequences substantially homologous thereto, and any fragment thereof; and a nucleic acid sequence which hybridizes to any one of the sequences described above. The nucleic acid that hybridizes under stringent conditions preferably has a sequence identity of about 70% with any of the sequences mentioned above, more preferably about 90%.
Another object of the invention is to provide a recombinant expression vector adapted for transformation of a host or for delivery of a sequence encoding OMP21 to a host, comprising the nucleic acid molecule of SEQ ID NO: 6, a complementary sequence thereof, sequences substantially homologous thereto, and any fragment thereof. Preferably, the recombinant expression vector is adapted for transformation of a host and comprises an expression means operatively coupled to the nucleic acid molecule for expression by the host of said OMP21. More preferred is the expression vector wherein the expression means includes a nucleic acid portion encoding a leader sequence for secretion or purification from the host of OMP21.
A further aspect of the invention includes a transformed host cell comprising an expression vector described above and OMP21 producible by the transformed host cell.
The invention further encompasses attenuated and/or inactivated cultivars of M. catarrhalis wherein the cultivar has been genetically manipulated to have the gene for OMP21 xe2x80x9cknocked-outxe2x80x9d and therefore non-transcribed (a xe2x80x9cdeletion-mutantxe2x80x9d), so that the adherence by the organism is reduced. Also encompassed in this invention are cultivars of M. catarrhalis having a double deletion of OMP21 and OMP106, as described in PCT application WO 97/41731, which is incorporated herein by reference in its entirety, so that the organism is non-adherent.
The invention further encompasses pharmaceutical compositions, including prophylactic and therapeutic compositions, and which may be immunogenic compositions including vaccines, wherein said immunogenic composition produces an immune response when administered to a host, comprising at least one component selected from the following group:
a) OMP21;
b) a nucleic acid molecule or a fragment or compliment thereof, encoding OMP21;
c) a nucleic acid molecule having the sequence of SEQ ID NO:6, the complimentary sequence thereto, a nucleic acid sequence which hybridizes under stringent conditions thereto, or fragments thereof;
d) OMP21, obtainable from a transformed host comprising an expression vector comprising a nucleic acid molecule as defined in b) or c) and expression means operatively coupled to the nucleic acid molecule for expression by the host of said OMP21;
e) a recombinant vector comprising a nucleic acid or fragment or analog thereof, encoding OMP21; and
f) a transformed cell comprising the vector of e),
optionally one or more adjuvants, and optionally a pharmaceutically acceptable carrier or diluent.
The invention further encompasses pharmaceutical compositions, including prophylactic and therapeutic compositions, and which may be immunogenic compositions including vaccines, comprising an attenuated and/or inactivated M. catarrhalis cultivar provided herein, optionally an adjuvant, and optionally a pharmaceutically acceptable carrier or diluent.
The invention further encompasses the pharmaceutical compositions described above, optionally in combination with, fused to, or conjugated to another component, which may be in an immunogen, and may include but is not limited to: a lipid, a phospholipid, a carbohydrate including a lipopolysaccharide, another protein, and an attenuated or inactivated whole organism, as provided herein or known to those skilled in the art, including but not limited to those described in Section 2 above. Preferred optional components include any Moraxella, Neisseria, Pseudomonas, Streptococcus, or Haemophilus attenuated or inactivated whole organism, or a protein or a carbohydrate therefrom. Preferred immunogenic compositions, including vaccines, comprise OMP21 in combination with OMP106 and one or more adjuvants.
Also included are methods for producing an immune response in an animal comprising administering to said animal an effective amount of an immunogenic composition described above.
Another aspect of the invention is directed to the antisera raised against any of the immunogenic compositions described above, and the antibodies present in the antisera that specifically bind the immunogens present in the immunogenic composition, including OMP21 or nucleic acid encoding same, and other immunogenic components.
The invention also includes diagnostic reagents which may include any of the above mentioned aspects, such as the isolated OMP21, the nucleic acid molecule encoding OMP21, the immunogenic composition, the antisera, the antibodies, the vector comprising the nucleic acid, and the transformed cell comprising the vector.
Methods and diagnostic kits for detecting OMP21, M. catarrhalis, anti-OMP21 antibodies or anti-M. catarrhalis antibodies in a test sample are also included, wherein the methods comprise the steps of:
a) contacting a test sample with an antigenic or immunogenic composition of the present invention or antibodies thereto to form antigen:antibody immunocomplexes, and further,
b) detecting any immunocomplexes formed during step a) as an indication of the presence of said antigen or antibodies in a test sample. The methods may further comprise quantitating any said immunocomplexes formed.
The diagnostic kits for detecting OMP21, M. catarrhalis, or antibodies thereto, comprise the antibodies and/or the antigenic or immunogenic composition of the present invention, a container means for contacting said antibodies or antigenic or immunogenic composition with a test sample suspected of having said antibodies and reagent means for measuring antigen:antibody immunocomplexes formed between said antigenic or immunogenic composition and said antibodies.
A further aspect of the present invention provides methods for determining the presence of nucleic acids encoding OMP21 in a sample, comprising the steps of:
a) contacting a sample with the nucleic acid molecule provided herein to produce duplexes comprising the nucleic acid molecule and any nucleic acid molecule encoding the OMP21 in the sample and specifically hybridizable therewith; and
b) determining the production of duplexes.
The present invention also provides a diagnostic kit and reagents therefor, for determining the presence of nucleic acid encoding OMP21 in a sample, comprising:
a) the nucleic acid molecule as provided herein;
b) means for contacting the nucleic acid with the sample to produce duplexes comprising the nucleic acid molecule and any nucleic acid molecule encoding the OMP21 in the sample and specifically hybridizable therewith; and
b) means for determining the production of duplexes.
Also included in this invention are methods of preventing, treating or ameliorating disorders related to M. catarrhalis in an animal, preferably a human, in need of such treatment comprising administering an effective amount of a pharmaceutical composition provided herein. Preferred disorders include a M. catarrhalis bacterial infection, otitis media, respiratory infections, sinusitis and pneumonia. Preferred vaccines and pharmaceutical compositions include those formulated for in vivo administration to an animal, preferably a human, to confer protection against disease, or treatment therefor, caused by a strain of M. catarrhalis. Also preferred are compositions formulated as a microparticle, capsule, or liposome preparation.
Nucleotide or nucleic acid sequences defined herein are represented by one-letter symbols for the bases as follows:
A (adenine)
C (cytosine)
G (guanine)
T (thymine)
U (uracil)
M (A or C)
R (A or G)
W (A or T/U)
S (C or G)
Y (C or T/U)
K (G or T/U)
V (A or C or G; not T/U)
H (A or C or T/U; not G)
D (A or G or T/U; not C)
B (C or G or T/U; not A)
N (A or C or G or T/U) or (unknown)
Peptide and polypeptide sequences defined herein are represented by one-letter or three-letter symbols for amino acid residues as follows:
The present invention may be more fully understood by reference to the following detailed description of the invention, non-limiting examples of specific embodiments of the invention and the appended figures.